Fluorescent molecules have been known for more than 100 years in the art and have become increasingly important for the detection of small molecule, biomolecule, polymer and other analytes when covalently or otherwise associated with such analytes. Fluorescent entities are now frequently used in high throughput drug discovery screening in the pharmaceutical, agrochemical, cosmetic, polymer and biotechnology industries and in many cases taking the place of the environmentally unfriendly and undesirable radioimmunoassay (RIA) that necessitates the controlled use and disposal of radioactive materials. The fluorescent assay often shows improved sensitivities over the equivalent RIA assay and is of course much safer and simpler for laboratory workers to execute. Examples of fluorescent techniques in these industries are; conventional fluorescence and luminescence assays, ELISA (Enzyme-linked immunosorbent assay) fluorescence polarization assays, fluorescence resonance energy transfer (FRET) assays and fluorescence activated cell sorting (FACS) procedures. Traditionally, fluorescent reporter molecules in the above applications have had several undesirable properties. Most dyes are very hydrophobic or contain bare sulfonate groups (Ar—SO3−) that are responsible for a high level of non-specific binding to proteins and peptides. Hydrophobic dyes or dyes containing bare aromatic sulfonate substituents form aggregates that are responsible for many undesirable effects such as self-quenching or absorbance emission shifts, Many traditional dyes such as fluorescein have limited pH dependent water solubilities and absorption/emission wavelengths in the 350-500 nm range of the electromagnetic spectrum. This is not the most convenient wavelength range with which to monitor biological systems as a host of biological molecules also absorb light in this region, for example, hemoglobin. At pH 7, the absorbance maximum of fluorescein occurs at 490 nm and emission is at around 515 nm. A much better range is 550-850 nm where the biological absorption window and autofluorescence is at a minimum. In the near-infrared region of the electromagnetic spectrum above 850 nm, water starts to absorb very strongly, dramatically lowering dye sensitivity. Also, traditional dyes such as fluorescein and rhodamine are less photostable than many of the longer wavelength absorbing dyes particularly when intense excitation is required such as in fluorescence microscopy, in part, due to their inherent “higher energy” absorption at shorter wavelengths.
Novel fluorescent dyes that operate in the visible/near infra-red (NIR) region of the electromagnetic spectrum have been shown to act as excellent reporter components of targeting molecular probes and tracers. However, many of the available dyes have undesirable properties such as relatively high non-specific binding and a tendency to aggregate. The main focus of this patent is the discovery of novel cyanine dyes for use in high throughput screening, fluorescence polarization, biomedical imaging and other applicable techniques such as immunofluorescence microscopy and fluorescence activated cell sorting and counting. The new dyes are designed to have much improved properties to function in biological systems, the main improvement being the use of more appropriate positively charged water solubilizing groups that improve sensitivity in proteinaceous assays and reduce dye aggregation. The new dyes are designed to absorb in the long visible near-infrared region between 550 and 850 nm. The dyes have high molar extinction coefficients of over 100,000 M−1cm−1; this is a measure of how efficiently the dyes absorb light of a particular wavelength, the higher the molar extinction coefficient, the higher the efficiency of light absorption. The bench standard, fluorescein, has an extinction coefficient of 80,000 M−1cm−1 above 6 in water or methanol. Extinction coefficients are routinely measured in the laboratory using UV/Visible spectrophotometers. Many of the biotechnological and pharmaceutical companies have an in-house chemical library of compounds, many of these compounds themselves absorb light up to around 600 nm where the commonly used shorter wavelength dyes such as fluorescein and rhodamine dyes operate making them much less efficient than a longer wavelength dye absorbing light above 600 nm.
Whole animal cellular and molecular imaging has the potential to dramatically accelerate drug discovery and development by revolutionizing in vivo research. As with all imaging techniques, animals do not have to be sacrificed at each data collection point and they can therefore serve as their own controls. Also, the instrumentation is relatively low cost, can be computerized so more data can be collected at more frequent time points. In 1995, Christopher H. Contag and co-workers described a method for noninvasive optical monitoring of microbial infections in a whole animal. Their model system began with infection of mice with strains of the bacterial pathogen Salmonella typhimurium which were modified to produce bioluminescence via the luciferin/luciferase mechanism. This was effected by constitutive expression of a luciferase enzyme from the soil bacterium Photorhabdus luminescens. Salmonella typhimurium is an intracellular pathogen of mice, humans and other animals which initially infects the intestinal mucosa after oral ingestion. It then spreads systemically largely by unknown mechanisms to many sites within the host. The authors used a modified near infrared CCD camera to detect light emission from the bacterium. They discovered that the highest intensity emission came from the caecum where the bacteria appeared to gather before assaulting other tissues. They went on to perform qualitative real time studies on the efficacy of administered antibiotics and witnessed rapid clearance of fully virulent bacterial strains by naturally bacterial resistant mouse strains. These initial experiments were reported to be somewhat hindered by the requirement of oxygen for the intracellular light producing reaction and more so by the short 486 nm wavelength emission of the bioluminescent system. Due to the short wavelength of the emitted light, much of the light was absorbed by the animal's own tissue and plasma components such as hemoglobin. Tumor imaging work using labeled tumor targeting antibodies confirmed that further red-shifted dyes were far superior to fluorescein for biomedical “whole body” imaging as they had lower backgrounds, better circulating lifetimes and were able to emit light that penetrated further through general tissue due to much less interference from the animal's own tissue and plasma components. The new cyanine dyes could be used as reporter groups in these kinds of experiments.
There are currently two types of whole body optical imaging, the first, bioluminescent imaging, is where a light emitting gene system is introduced into the animal such as the luciferase/luciferin system or green/red fluorescent proteins. The second type is where a fluorescent tracer molecule is used and external light is required. The first emits its own light but requires a plentiful supply of oxygen and cannot be used above 600 nm. This is a severe limitation as much of the light is lost due to poor tissue penetration as described above. The second “red-shifted” fluorescent tracer method is increasingly being used and emitted light can easily penetrate to tissue depths sufficient for good clinical imaging in small rodents such as mice. Developments in imaging technologies have had a profound impact on clinical medicine including, ultrasound scanning, magnetic resonance imaging, x-ray computed tomography and nuclear tomography imaging. These systems are primarily used for displaying anatomical, physiological and metabolic parameters but they are increasingly being used in experimental animal systems for imaging at the cellular and molecular levels in vivo. These currently used imaging systems are well developed but rely on physical parameters and properties to generate image contrast such as, sound impedance, electromagnetic wave impedance or nuclear alignments. Methods that involve X-rays are also inherently unsafe in that prolonged exposure to X-rays is known to cause cancer. In vivo fluorescence imaging offers very safe, more sensitive early stage detection of tumors or enzymes that would be difficult to achieve with existing imaging techniques.15 
Fluorescent tracer molecules are commonly used as tools for in vivo imaging. The tracer molecule is composed of two parts, a targeting moiety which is typically an antibody, peptide or DNA oligomer, and a reporting component which, in fluorescent imaging, is a fluorescent molecule. For tumor imaging for example, the targeting component could be an antibody or peptide that is known to bind more specifically to the tumor cells than to normal host cells. Tumor cells often over-express certain surface proteins or particular receptors that targeting antibodies can be raised against. Certain cancers such as head, neck and oral cancers are known to greatly over-express the EGF receptor, a 170 kDa glycoprotein with tyrosine kinase activity and again, antibodies or peptides have been used to target these tumors. Weissleder recently introduced new targeting concepts by developing protease activated near-infrared fluorescent probes for tumor detection and for receptor imaging using a targeting fluorescent peptide.
A variety of other techniques would benefit from the discovery of efficient long wavelength, near-infrared dyes. For example, immunofluorescence microscopy is an indispensable tool in cell biology, microbiology and histology. It provides for specific visualization of a particular protein or other biological target of interest in live or fixed cells while all other biomolecules remain invisible. Traditionally, the shorter wavelength dyes fluorescein and rhodamines (490-590 nm) have been used in this area but autofluorescence from unlabeled biomatter significantly lowers these probes sensitivity. Paraformaldehyde fixing of tissues also causes an increase in the inherent tissue autofluorescence in this range. Another use for the dyes being made in this project is in the area of fluorescence activated cell sorting or FACS. FACS machines have been used for some time to rapidly quantify molecular events in cells. This technique has also been used in screening solid phase chemical libraries where the fluorescently labeled antibodies or enzymes bind to resin bound test peptides or small molecules on the bead surface. Many new whole cell assay procedures are now being developed on these machines where the cells or beads are just being counted, not sorted. The fluorescent event, which could be either a simple receptor or antibody binding event or it could be a fluorescent enzyme assay, either occurs in the cell or does not occur and is rapidly detected and counted by the FACS machine. This field has undoubtedly been driven by the incredibly low cost of some the new cell counting machines. This patent describes a series of novel long wavelength dyes for potential use in the above applications.
The prior art recites cyanine dyes both with and without sulfonic acid or sulfonate groups (SO3H and SO3−, where negatively charged sulfonate groups are used both as water solubilizing groups and for the purpose of enhancing the fluorescence properties of the dyes see Waggoner U.S. Pat. No. 5,268,486, Ahlem in U.S. Pat. No. 5,955,612 and WO09641174 and Terpetschnig in U.S. Pat. No. 6,538,129 B1 and U.S. Pat. No. 7,250,517 B2 and references cited therein).